The present invention concerns an individual system for determining whole blood coagulation time, a disposable sensor of small dimensions including a specific thrombin enzyme reagent allowing an electrochemical determination and an electronic measuring apparatus allowing an electric signal received from said sensor to be correlated when it has been introduced into the apparatus and a drop of the blood to be analysed has been deposited thereon.
The invention concerns more particularly a measuring apparatus and sensor of this type allowing the prothrombin time (PT) to be determined by amperometry when the reagent composition deposited on the sensor includes a chemical substrate of which a terminal fraction can be selectively cut off by the thrombin enzyme by releasing a charged group.
Checking the blood coagulation time, i.e. the aptitude of the blood constituents to form a clot to prevent the risk of haemorrhage, forms part of the routine examinations, or even daily examinations, performed in numerous acquired pathological, traumatic or postoperative situations. It is for example necessary during treatment with anticoagulants in the case of heart related diseases to be able to adjust the dosage of the medicine accurately, for example warfarin or heparine, in order to avoid the risk of haemorrhages in the event of an overdose or conversely, the risk of thrombosis if the quantity of anticoagulant administered is insufficient.
Different parameters have been retained for performing this determination, but the most common is measuring the prothrombin time (PT) after activation, i.e. the period of time after which the formation of a clot is observed with a blood sample taken from the patient. Such analyses were for a long time entirely dependent upon the expertise of specialised laboratory personnel equipped with complex and cumbersome apparatus. This had the drawback of obliging the patient to travel, requiring preparation of a blood sample taken at home, for example by adding citrate, to wait until a laboratory analysis could be performed.
Progress made as regards miniaturisation, in particular using electronic components, has, for slightly more than ten years or so, enabled the patient to have various types of more compact equipment allowing him to perform a coagulation time measurement at home. Most of these individual pieces of equipment rely on the same principles as laboratory apparatus, namely direct observation of the erythrocyte dynamics in a blood sample, to which the usual coagulation reagents have been added, when it changes from a fluid state to a viscous or clotted state.
According to a first principle, the period of time after which a prepared blood sample no longer flows through a capillary tube, or through the calibrated choke of a tube of larger diameter, supported by single use receiving means which can be fitted to a measuring apparatus, is measured. This flow is generally forced by means of a pneumatic pump device integrated in the measuring apparatus, and the period of time after which coagulation occurs is generally detected by optical means. Devices of this type are for example disclosed in U.S. Pat. Nos. 3,486,859, 3,890,098 and 5,302,348. An apparatus relying on this principle is for example that proposed under the trademark  less than  less than Hemochron greater than  greater than  or according to a more recent variant under the trademark  less than  less than Protime Microcoagulation System greater than  greater than  by International Technidyne Corporation (NJ-USA). The measuring apparatus obviously includes a power source for supplying power both to the mechanical part (pump) and the end of coagulation check (optical detection). It will further be observed that each receiving means for the blood sample to be analysed, which is disposable after the first use is relatively cumbersome (approximately 3xc3x979 cm) and depends upon a precision technology (calibration of the capillary tube or the choke) which necessarily contributes towards increasing the cost of each analysis performed.
According to a second principle, one measures the period of time after which a prepared blood sample, deposited in a disposable cupel allows the immobilisation by coagulation of a magnetic object moved by a rotating magnetic field, the detection of the coagulation phenomenon being again most often performed by optical means. U.S. Pat. No. 3,967,934 already discloses this principle wherein the container intended to receive the sample contains a ferromagnetic ball. Such as device is for example used in the apparatus distributed by Nycomed Pharma (Oslo, Norway) under the trademark  less than  less than Thrombotrack greater than  greater than . According to another variant, described for example in U.S. Pat. No. 5,154,082, the ball is replaced by ferromagnetic particles, which are also subjected to an electromagnetic field. This has enabled a reagent to be made in a dry form, deposited on a support, the mobility of the erythrocytes still being detected by optical means. A device of the preceding type corresponds for example to a Boehringer Mannheim (Germany) apparatus sold under the trademark Coaguchek. The products proposed corresponding to this second principle have the same drawbacks as those already mentioned for the apparatus according to the first principle with the exception perhaps of the lower cost of the blood sample receiving means for the Coaguchek apparatus.
Comparative tests performed with the methods and devices of the aforecited prior art ( less than  less than Home Prothrombin Estimation greater than  greater than  by Angelida Bernado et al., Thrombosis, Embolism and Bleeding, ch. 3.5xe2x80x94E.g. Butchart and E. Bodnar ICR Publishers 1992) have demonstrated that the medical follow-up of a patient at home was at least as satisfactory as that of a patient in a hospital environment, but that reliable and reproducible results could not be obtained unless the patient had had a reasonable period of training. The apparatus of the prior art are of course provided for domestic use, can easily be moved but yet remain relatively too voluminous for a patient to be able to keep at home about his person, for example in a pocket, while he moves around. It is doubtless also desirable, for the reliability of the measurements which depend upon devices which are both optical and electromechanical, for said apparatus to be moved as little as possible.
It will be observed finally that, according to one or other of the above principles, the electric power source necessary for supplying power to the optical and electromechanical devices must be relatively large when it is autonomous (battery) but that it is never directly involved in the coagulation time measurement.
Reference can however be made to a U.S. Pat. No. 3,674,014 of 1972 which discloses a syringe whose inner wall includes a succession of electrodes allowing the variation in impedance of the analyte to be measured, by means of an oscilloscope connected to said syringe, progressively as the coagulation phenomenon occurs. Such a device still only relies on the variation in the properties of the analyte during coagulation and is evidently not intended for individual use.
The device disclosed in European Patent No. EP 0 679 193, allowing inter alia, the prothrombin time to be measured, includes two electrodes which are only involved in said determination to detect, from a signal representing the resistance variation between the electrodes, the presence of a blood sample on the receiving means, and have no direct role in measuring a period of time. In this device, the period of time measurement is performed by a photometric determination of the fluorescence of the medium from an oligopeptidic substrate having Rhodamine as its leaving group able to be released by being cut off by the thrombin enzyme. A substrate of this type is for example disclosed in U.S. Pat. No. 4,557,862. A colorimetric method using a slightly different substrate, having p-nitroaniline as chromatophore, corresponds for example to the product marketed by Nycomed Pharma under the trademark  less than  less than NYCOTEST-CHROM greater than  greater than .
These calorimetric methods have the advantage of no longer relying on mechanical or electromechanical means, but, on the other hand, they require an additional centrifugation or ultrafiltration step through a membrane, as proposed in European Patent No. EP 0 679 193, to remove the erythrocytes in order to perform detection on the plasmatic part alone. These methods have the additional drawback of requiring a measuring apparatus having high precision optical detection means, thus a relatively fragile apparatus which is difficult to transport, and requiring a relatively long colour development time, generally longer than 5 minutes, before the measurement can be performed.
A method with a simpler principle, which relies on an electrochemical measurement, is disclosed in U.S. Pat. No. 4,304,853. This method consists in introducing into a measuring cell including at least two electrodes, a citrated blood sample and an oligopeptidic substrate which has to be kept in a solvent medium such as dimethylsulfoxide (DMSO). The substrate is formed of a chain of amino-acids having arginine as the terminal amino-acid, linked to an electroactive leaving group and of which one hydrogen of the initial amino-acid can be replaced by a protective group, these amino-acids, leaving groups and protective groups being selected from very limited lists. Because of the complexity of the coagulation phenomenon due to a reaction cascade, the presence of a solvent can interfere with the intermediate products leading to a release of thrombin and thus prejudicing the accuracy of the measurement.
In an international PCT Patent Application entitled  less than  less than Oligopeptidderivative greater than  greater than  filed on the same day by the company Pentapharm AG (Basel, Switzerland), a particular selection of the constituent links of the substrate, in particular of the amino-acids and leaving group, allow determination of the coagulation time of a blood sample in a liquid medium to be performed, using a measuring cell of the same type as used previously, without it being necessary to add a solvent or a co-solvant to keep the substrate in solution. Even if the use of these new substrates has a certain advantage, the use of a measuring cell, with the handling which this requires, still does not provide a patient, who would like to check his coagulation time himself, with an apparatus which is easy to transport and simple to use.
The object of the system according to the invention is to overcome the drawbacks which still exist in the systems of the prior art by providing an apparatus and sensors of much smaller size as a result of the use of specific reagents and a different methodology to that previously used for determining an individual coagulation time.
The invention thus concerns an electrochemical system for measuring a value representing the coagulation time of a drop of whole blood characterised in that it includes:
an electrochemical sensor in the shape of a strip of small dimensions bearing at least a reference electrode and a working electrode on which a specific reagent is immobilised, the composition of said reagent incorporating at least one chemical substrate one terminal link of which can be cut off by the thrombin enzyme to give charged groups (LG), and
a measuring apparatus whose electronic circuit, which is supplied with power by a power source, allowing a variable or non variable electric current or a variable or non variable potential difference to be imposed across the electrodes of the sensor, the electric signal resulting from the migration of the charged groups to be processed, said signal to be correlated with a value representing the coagulation time, and said value to be displayed on a display panel of said measuring apparatus.
The specific reagent immobilised on the working electrode further includes a thromboplastin and a buffer medium.
The chemical substrate one terminal link of which can be cut off by the thrombin enzyme to release charged groups LG must have a stereospecific site for the thrombin enzyme. An appropriate chemical substrate is for example formed by an oligopeptidic derivative and more particularly an oligopeptidic derivative having arginine (arg) as the terminal amino-acid linked to a group able to be cut off by thrombin to give charged groups (LG). According to a preferred embodiment, these groups which can be released by thrombin are selected from the amino-aryl or amino-heteroaryl groups which may be substituted, the link with arginine being performed by one of their amine functions.